Various tumors express oligosaccharide sequences which are different from the non-malignant glycosylation of the same cell or tissue type. Examples of the known or speculated cancer associated oligosaccharide structures include: glycolipid structures such as globo-H (Fucα2Galβ3GalNAcβ3Galα4LacβCer), gangliosides: GM1 Galβ3GalNAcβ4(NeuNAcα3)LacβCer or GD2 GalNAcβ4(NeuNAcα8NeuNAcα3)LacβCer; Lewis-type fucosylated structures such as Lewis a and x: Galβ3/4(Fucα4/3)GlcNAc, Lewis y: Fucα2Galβ4(Fucα3)GlcNAc, sialyl-Lewis x: NeuNAcα3Galβ4(Fucα3)GlcNAc, and some combinations of these on polylactosamine chains; O-glycan core structures, such as T-antigen Galβ3GalNAcαSer/Thr-Protein, Tn-antigen GalNAcαSer/Thr-Protein or sialyl Tn-antigen NeuNAcα6GalNAcαSer/Thr-Protein. Presence of non-human structures such as N-glycolyl-neuraminic acid in cancers has also been indicated. Association and specificity of oligosaccharide structures with regard to cancers have been well established only in few cases, some of the structures are present in normal cells and tissues and are possibly only more concentrated in cancers.
One report has indicated that structures with terminal GlcNAcβ3GalβGlcNAc sequence are present in human leukemia cells (Hu et al., 1994). The structures may also be equally present on normal leukocytes. Thus, the relation of the finding to glycosylation patterns generally present in solid tumors was not indicated. This type of saccharide structures may be a part of rare normal glycosylations of human tissues: GlcNAcβ3Galβ4GlcNAcβ6sequence linked on O-glycans is probably present on human gastric mucin. A study shows that a monoclonal antibody recognizing GlcNAcβ3Galβ4GlcNAcβ6 sequence may possibly recognize similar structures on malignant tissues, such as mucinous ovarian neoplasms, pseudopyloric metaplasia of gallbladder and pancreatic epithelia, gastric differentiated carcinoma of stomach, gallbladder and pancreas, and on non-malignant tissues, such as human amniotic fluid, but, however, the structures from malignant tissues were not characterized (Hanisch et al., 1993). The antibody did not recognize neoglycolipid structure GlcNAcβ3Galβ4GlcNAcβ3Galβ4 nor carcinomas of lung, colorectum, endometrium or other organs. Another monoclonal antibody raised against testicular cells probably recognizes branched N-acetyllactosamines such as GlcNAcβ3(GlcNAcβ6)Galβ4GlcNAc- (Symington et al., 1984). Terminal GlcNAc has also been reported from mucins of human foetal mucin (Hounsell et al., 1989). In normal tissues terminal GlcNAc may be present in minor amounts as biosynthetic intermediates in the biosynthesis of poly-N-acetyllactosamines.
Several monoclonal antibodies has been raised against a semisynthetic glycolipid GlcNAcβ3Galβ4GlcNAcβ3LacβCer, these antibodies were shown to recognize glycolipids from cultured colon cancer cell lines and tumors (Holmes et al., 1991). However, the antibodies recognized several structures and the binding data was contradictory. Moreover the glycolipids were not recognized by all of the antibodies and the glycolipid structures from cancer cells or tumors were not characterized. Therefore the presence of terminal GlcNAc structures on tumors were not established. Another study showed production of a monoclonal antibody against GlcNAcβ3LacβCer (Nakamura et al., 1993). This antibody also weakly recognized the pentasaccharide structure described above. Moreover, the antibody recognized a protease sensitive epitope on COS-1 cells, which cell line is not of human origin. The immunization protocols of these studies did not describe induced antibody responses against polyvalent conjugates of the saccharides, but immunization by glycolipids.
Normally there are large amounts of antibodies recognizing terminal GlcNAc structures in human serum. There is also a class of natural antibodies recognizing terminal Galα3Galβ4GlcNAc- structures. The Galα antigen is not naturally present in man and recently it was also shown that the natural antibodies bind structures such as GalNAcα3Galβ4GlcNAc, GalNAcβ3Galβ4GlcNAc, and GlcNAcβ3Galβ4GlcNAc (Teneberg et al., 1996). The X2-structure, GalNAcβ3Galβ4GlcNAc, is a normal antigen on human tissues and structures GalNAcα3Galβ4GlcNAc and Galα3Galβ4GlcNAc have not been described from normal or cancer tissues. Thus, the present finding that the terminal GlcNAc structure is a tumor antigen indicates that the actual function of the natural antibodies might be the prevention of cancers having terminal GlcNAc structures.
The following patents describe cancer antigens and their use for making antibodies for therapeutic and diagnostic uses and for cancer vaccines. The antigen structures are not related to saccharides of the present invention:
Cancer vaccines: U.S. Pat. Nos. 5,102,663; 5,660,834; 5,747,048; 5,229,289 and 6,083,929.
Therapeutic antibodies: U.S. Pat. Nos. 4,851,511; 4,904,596; 5,874,060; 6,025,481 and 5,795,961.
Diagnostics: U.S. Pat. Nos. 4,725,557; 5,059,520; 5,171,667; 5,173,292; 6,090,789; 5,708,163; 5,679,769; 5,543,505; 5,902,725 and 6,203,999.
In the prior art tumor diagnostic and therapeutic antibodies recognizing chitobiose-mannose trisaccharides has been described in DE 38 07 594 A1. The application also describes other N-glycans with numerous varying terminal structures some of which may comprise also non-reducing terminal N-acetyl glucosamine. Several of the desired structures have been characterized as normal glycans and not cancer specific structures. The application claims to describe structures useful for cancer applications. However, it is not clear from the invention what the structure of the desired glycan is. Formel (I) may indicate presence of non-reducing terminal GlcNAc, if it is unconventionally read from right to left. However the Formel (I) does not indicate the linkage structure of the terminal GlcNAc. The Formel (III) indicates that the GlcNAc residues are α2, α4, or α6-linked. The present invention is not directed to such unusual structures. The present invention is directed to human tumor specific glycans comprising non-reducing end terminal β-linked GlcNAc residues.
Patent application WO 00/21552 claims several unusual O-glycan structures isolated from bovine submaxillary mucin. Two of the structures such as GlcNAcβ6GalNAcα6GalNAc and GalNAcβ3(GlcNAcβ6)GalNAc comprise terminal GlcNAc-residues. The application did not indicate that said structures would also be related to bovine or human cancers. The present invention is not directed to these structures comprising two GalNAc-residues. The application contains speculation about potential therapeutic use of the structures as antigens related to cancer. However, it has not been shown that the structures are related to bovine cancer when these are present in bovine normal submaxillary secretion. Moreover, it is even less probable that the structures would be present in human tissues, the glycosylations are species specific and vary between human and bovine, e.g. bovine and human glycosyltransferase and glycosylation profiles are different. The human genome is also known and thus gylcosyltransferases which could be related to synthesis of the claimed bovine structures should have been now produced and characterized from human. So far none of these has been described in human, or human cancer.
Meichenin et al, 2000 shows a murine monoclonal antibody which can bind to some oligosaccharides containing terminal GlcNAcβ and to certain human tissue cancer samples. The antibody was produced in mouse using the endo-beta-galactosidase treated red cells. However, the article does not establish any human specific immunotherapy with a specific antibody or other carbohydrate specific binding substance binding to an immunogenic carbohydrate. Moreover, the fact that such an antibody with limited specificity was formed in mouse does not indicate that similar antibody would be formed in human, or it would be tolerable in human, or be useful in context of individual human tumors of specific kind, since immune reactions to carbohydrates are species specific. Endo et al, 1996 describes N-glycan type oligosaccharides from soluble protein alkaline phosphatase of rat hepatoma AH-130 cell line. Again, material of animal origin is speculated in view of cancer specificity. Therefore, no diagnostics or therapy of human cancer is disclosed.
Patent application FI20011671 described the general usability of terminal GlcNAc-structures in tumor therapy. The application described specific polylactosamine type oligosaccharide sequences containing terminal GlcNAc linked to Gal especially found from glycolipid. The application indicated that the structures can be part of N-linked glycan or O-linked glycans, and that the tumorspecific oligosaccharide sequences can also be linked to O-glycosidic GalNAc. The application did not disclose exact structures of the O-linked or N -linked glycans.
The present invention describes preferred treatment of cancer when the oligosaccharide sequences have been detected from cancer but not from normal tissues. The present invention is directed to the combination of the analysis and treatment of a cancer. The present application also show for the first time the usability of the oligosaccharide sequences of the terminal beta-GlcNAc sequences for cancers of lung, stomach, colon, larynx and mucinous carcinomas, especially mucinous ovarian carcinomas, forming a group of epithelial type and/or mucin secreting cancers. The present invention is further especially directed to human cancer specific protein linked GlcNAcβ-structures. The present invention is also further especially directed to the specified N-glycans and O-glycans and protein linked GlcNAc. The preferred structures form a specific family of terminal specifically “protein linked GlcNAcβ-structures” which are human protein linked GlcNAc O-glycans and N-glycans form a specific family of human cancer specific “protein linked GlcNAcβ-glycan cores” which are result of defective galactosylation of cancer or tumor tissue.
The present application further describes human natural antibodies and more specifically human cancer associated antibodies specifically recognizing preferred human terminal beta-linked GlcNAc structures. The present invention also describes an enzyme based targeting of the cancer antigens by transferring a modified monosaccharide derivative on cancer cells. The presence of the human natural cancer associated antibodies shows that it is possible to use the structures as targets for cancer therapy in human in vivo. The invention is specifically further directed to antibody and other cancer targeting therapies and therapeutic immune reactions such as cancer vaccination and reagents useful for these directed to the preferred specifically protein linked GlcNAcβ-structures. The present invention is specifically further directed to antibody and other cancer targeting therapies and therapeutic immune reactions such as cancer vaccination and reagents useful for these directed to human cancer specific protein linked GlcNAcβ-glycan cores.
Current therapies for cancer and numerous infectious diseases are not effective enough. Cancers are major cause of deaths in industrialized countries and devastating infectious diseases kill children and adults especially in developing countries. Infections are probably behind numerous life-style and other diseases of the industrialized world, like gastric ulcers caused by Helicobacter pylori. 
Previous in vitro studies have described the transfer of sialyl-Lewis x-oligosaccharides on surface of a cultured cell type. The cells were used to study the binding of human selectins to the sialyl- Lewis x oligosaccharides. Similarily, a blood group B-antigen has been transferred to human erythrocytes, and it was shown to be recognized by anti-blood group B antibodies. Because of the unnatural structure of the GDP-Fuc(-B-antigen), the authors stated that the structure was unsuitable for in vivo use. According to present invention, it is possible to use the unnatural structures as modified monosaccharides to target microbial pathogens, viruses, tumors or cancers.
A galactosyltransferase has been used to label human endo-beta-galactosidase modified erythrocytes (Viitala and Finne, 1984) and mouse teratocarcinoma cells (Spillmann and Finne, 1994) under in vitro conditions. Cell surface galactosyltransferases has been also studied in connection of various biological conditions. These studies do not describe diagnostics or therapy for any disease. The carbohydrates transferred are not monosaccharide conjugates according to the invention. The old studies utilized radioactively labeled UDP-Gal, the chemical structure of the Gal-residue is not changed but it contains atoms enriched with 14C or 3H.
A fluorecently labelled muramic acid has been transferred on bacteria and the method was speculated to be used to study vaccination against the compound transferred or for interaction studies between bacteria, the transfer reaction was not specific for a type of a bacterium. The method was aimed to be used in vitro and the cells had to be permeabilized to achieve very weak reactions by the peptidoglycan precursor molecules (Sadamoto, R. et al 2001). In contrast the transfer reactions described by the present invention for bacteria are targeted to transfer of the carbohydrates directly to the cell surface. Preferentially the present invention is directed to in vivo use in the patient, which can be a human patient. Preferentially the invention is directed to use of transferring enzymes from the patients serum or transferring enzyme on the very surface of the pathogenic entity. As a separate embodiment the present invention is directed to direct cell surface vaccination against the pathogenic entity when a carbohydrate epitope is transferred.
Moreover the previous technology describes in vitro transfer of Gal on acceptors of a cancer cell by α1-3galactosyltransferase. The monosaccharide to be transferred is not modified nor aimed for blocking of a pathogenesis-inducing carbohydrate receptor. The invention does not describe transfer of immunologically active or toxic monosaccharide conjugates, but the terminal structure formed is reactive to anti Galα1-3Gal-antibodies. The epitope was aimed for increasing immununoreactivity of cancer cells to be injected as cancer vaccine.
Previous art also described transfer of 6-biotinylated Gal to GlcNAc-BSA or hen egg white ovalbumin or transfer of fluoresceinyl-NeuNAc to glycoproteins of golgi apparathus. The studies did not describe the therapy or diagnostics according to the invention (Bulter T. et al. 2001).
Various cell types express oligosaccharide sequences which are different from the non-malignant glycosylation of the same cell or tissue type. Association and specificity of oligosaccharide structures with regard to cancers have been well established only in few cases, some of the structures are present in normal cells and tissues and are possibly only more concentrated in cancers. The present patent application also describes terminal N-acetylglucosamine containing tumor specific oligosaccharide sequences. Priority of the recognition of the tumor specific oligosaccharide sequences by glycosyltransferases is claimed from the patent application.
Normally there are large amounts of antibodies recogniting terminal GlcNAc structures in human serum. Thus, the previous finding that the terminal GlcNAc structure is a tumor antigen indicates that the actual function of the natural antibodies might be the prevention of cancers having terminal GlcNAc structures.